We are using mutant enzymes obtained by recombinant DNA technology to examine the relationship between the structural and kinetic properties of DNA polymerases and their fidelity. The fidelity of exonuclease-deficient Klenow polymerase has been examined as a function of reaction pH. Overall fidelity increase more than 10-fold as the pH is lowered from 7.6 to 6.2, but rates are not affected equally for all errors. These non-random effects are consistent with steady-state kinetic analyses indicating that lowering the pH lowers the efficiency of mispair extension synthesis. Variations in the termination probabilities of more than 20-fold were observed at individual sites along the template DNA as a function of pH, and increased termination correlated with increased fidelity. These data suggest that high fidelity at low pH may result from both decreased formation of premutational intermediates due to the increased processivity of the polymerase and from decreased fixation of mutations due to decreased extension of misaligned or mispaired intermediates. We also extended the analysis to two new mutants in which a tyrosine residue at position 766, in the "O helix" of the polymerase active site, was changed to an alanine or serine. Both mutants have lower fidelity, suggesting that the tyrosine residue, known to bind dNTP substrates, is important for determining base selectivity. We are in the process of defining the detailed error specificity of these altered polymerases. We are also examining mutant derivatives of several other recombinant DNA polymerases, including HIV-1 reverse transcriptase, DNA polymerase beta, T7 DNA polymerase and T4 DNA polymerase. Analyses of these enzymes, for which considerable structural and/or kinetic data are available, should improve our understanding of accurate DNA synthesis and how fidelity is affected by DNA adducts.